Tumor necrosis factor (TNF) is a multifunctional cytokine that may play an important role in the pathogenesis of airway inflammation in asthma. TNF bioactivity is regulated by soluble TNF receptors that serve as TNF binding proteins. The specific aim of this project is to identify new mechanisms by which the release of soluble TNFR is regulated. We hypothesized that the mechanism of TNFR1 shedding might involve interactions with regulatory ectoproteins. Utilizing a yeast two-hybrid approach, we identified ARTS-1 (Aminopeptidase Regulator of TNFR1 Shedding) as a type II integral membrane protein that binds to the TNFR1 extracellular domain. In vivo binding of membrane-associated ARTS-1 to TNFR1 was confirmed by co-immunoprecipitation experiments utilizing human pulmonary epithelial and umbilical vein endothelial cells. A direct relationship exists between membrane-associated ARTS-1 protein levels and concordant changes in TNFR1 shedding. Cells over-expressing ARTS-1 demonstrated increased TNFR1 shedding and decreased membrane-associated TNFR1, while cells expressing anti-sense ARTS-1 mRNA demonstrated decreased membrane-associated ARTS-1, decreased TNFR1 shedding and increased membrane-associated TNFR1. ARTS-1 neither bound to TNFR2 nor altered its shedding, suggesting specificity for TNFR1. Although a GST-ARTS-1 fusion protein demonstrated selective aminopeptidase activity towards non-polar amino acids, multiple lines of negative evidence suggest that ARTS-1 does not possess TNFR1 sheddase activity. These findings indicate that ARTS-1 is a multi-functional ectoprotein capable of binding to and promoting TNFR1 shedding. Therefore, we have proposed that formation of a TNFR1-ARTS-1 molecular complex represents a novel mechanism by which TNFR1 shedding is regulated. Since both release of both soluble TNFR1 and IL-6 receptor-alpha (IL-6R) can be inhibited by hydroxamic acid based zinc metalloprotease inhibitors, we hypothesized that ARTS-1 might also regulate IL-6R shedding. Reciprocal co-immunoprecipitation experiments identified that membrane-associated ARTS-1 directly binds to a 55-kDa IL-6R, a size consistent with soluble IL-6R generated by ectodomain cleavage of the membrane-bound receptor. Furthermore, ARTS-1 promoted IL-6R shedding, as demonstrated by a direct correlation between increased membrane-associated ARTS-1 protein, increased IL-6R shedding, and decreased membrane-associated IL-6R in cell lines overexpressing ARTS-1. The absence of basal IL-6R shedding from ARTS-1 knock-out cells identified that ARTS-1 was required for constitutive IL-6R shedding. Furthermore, the mechanism of constitutive IL-6R shedding requires ARTS-1 catalytic activity. Thus, ARTS-1 promotes the shedding of two cytokine receptor superfamilies, the type I cytokine receptor superfamily (IL-6R) and the TNF receptor superfamily (TNFR1). We propose that ARTS-1 is a multi-functional aminopeptidase that may modulate inflammatory events by promoting the release of soluble receptors from at least two cytokine receptor superfamilies. Therefore, ARTS-1 may play an important role in regulating inflammatory events in the airway. Ongoing investigations will be aimed at identifying the mechanism by which ARTS-1 promotes cytokine receptor shedding.